Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), and malaria, caused by Plasmodium falciparum, remain amongst the world's deadliest infectious diseases. Co-infection with other diseases such as HIV plus the emergence of many drug-resistant strains worldwide have made these infections difficult and costly to treat. New drugs are needed that will kill wild-type and drug-resistant strains of both organisms. The major challenge in developing new antimicrobial agents is to identify metabolic processes that are both required for viability and able to be targeted by small molecules. The overall goal of our work is to discover and develop novel, potent antitubercular and antimalarial agents. We will achieve this by coupling the synthesis of potent small molecule inhibitors acting on-target intracellularly with downstream pharmacokinetic and animal experiments. This proposal centers on 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) as an antimicrobial drug target. Dxr is the first committed, and a rate-limiting step in the methylerythritol phosphate (MEP, aka nonmevalonate) pathway of isoprenoid biosynthesis. Dxr and MEP are essential for Mtb and P. falciparum survival, and the pathway is absent in humans. Current antimicrobial drugs do not work through a Dxr (or MEP) mechanism. Development of Dxr inhibitors as lead compounds against TB and malaria would be therapeutically valuable. Our prior work has resulted in several compound series that potently inhibit Dxr, kill both Mtb and P. falciparum-infected cells, act on-target intracellularly, and kill Plasmodium infection in mice. The proposed experiments are designed to further improve the efficacy of our compounds, verify the intracellular effects of Dxr inhibition, and evaluate the therapeutic potential of the most potent inhibitors. First, based on the success in our prior work, we will synthesize a series of novel, rationally-designed phosphonic acids. To improve cell penetration, lipophilic prodrug esters will also be synthesized. Second, compounds will be assessed for inhibition and mode of binding against purified recombinant Dxr from Mtb and P. falciparum. Third, we will measure the antimicrobial activity of our compounds against wild-type and drug-resistant strains. We will confirm the intracellular, on-target effects of the compounds. The most promising compounds will be evaluated in pharmacokinetics (PK) and animal efficacy assays. Overall, the experiments outlined in this proposal will result in potent antimicrobial compounds against both Mtb and P. falciparum and may provide a platform for further lead molecule development.